The plant hormone auxin controls diverse mitogenic and morphogenic events during plant development. Auxin signal- transduction regulates endogenous patterning processes, including early steps in meristem partitioning and formation of the plant vascular system. Auxin also regulates tropic responses to external stimuli such as light and gravity. Genetic screens have identified genes involved in auxin transport, auxin regulated gene transcription, and targeted protein degradation as critical elements in auxin signaling. However, the mechanisms by which these various components interact and are coordinately regulated is not yet known. The research proposed here is based on our recent isolation and characterization of the PINOID (PID) gene of Arabidopsis, that encodes a serine-threonine protein kinase. The pleiotropic PID loss-of-function and over-expression phenotypes resemble those of known auxin signaling and transport mutants, consistent with a specific role for this protein in auxin regulation. PID is the first kinase associated with auxin specific phenotypes. Therefore, PID offers a unique and powerful tool to explore the effects of protein phosphorylation on auxin- mediated processes. Analysis of the PID sequence indicates that PID is a member of a novel class of plant serine-threonine kinases. We will utilized genetic, molecular and biochemical strategies to initiate a detailed dissection of the role of PID and its homologues in the regulation of auxin activity in Arabidopsis. The primary aims of this study are (1) to use a genetic approach to identify pathway components that function upstream or downstream of PID in auxin signaling; (2) to use two- hybrid analysis to identify proteins that interact directly with PID; (3) to determine whether other PID-like genes in Arabidopsis also regulate auxin activity; and (4) to quantitatively measure the effects of PID mis-expression on global gene expression and auxin distribution. We will use in vitro and in vivo approaches to characterize candidate interacting genes and proteins and determine the specificity of the interactions. The proposed experiments will contribute to a fundamental understanding of the mechanisms behind auxin-mediated signal-transduction. This represents the first step toward the long-term objective of elucidating how endogenous and environmental signals interact with plant hormones to elicit specific developmental responses and to relate these mechanisms to signaling processes in other eukaryotic organisms.